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An in vitro screening cascade to identify neuroprotective antioxidants in ALS.

Barber SC, Higginbottom A, Mead RJ, Barber S, Shaw PJ - Free Radic. Biol. Med. (2009)

Bottom Line: Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system.These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated.Subsequent in vivo studies can be targeted using molecules with the greatest probability of success.

View Article: PubMed Central - PubMed

Affiliation: Academic Neurology Unit and Sheffield Care and Research Centre for Motor Neuron Disorders, University of Sheffield, Sheffield, UK.

ABSTRACT
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, characterized by progressive dysfunction and death of motor neurons. Although evidence for oxidative stress in ALS pathogenesis is well described, antioxidants have generally shown poor efficacy in animal models and human clinical trials. We have developed an in vitro screening cascade to identify antioxidant molecules capable of rescuing NSC34 motor neuron cells expressing an ALS-associated mutation of superoxide dismutase 1. We have tested known antioxidants and screened a library of 2000 small molecules. The library screen identified 164 antioxidant molecules, which were refined to the 9 most promising molecules in subsequent experiments. Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system. The top-performing molecules identified include caffeic acid phenethyl ester, esculetin, and resveratrol. These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated. Subsequent in vivo studies can be targeted using molecules with the greatest probability of success.

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Oxidative stress can be induced in NSC34 cells by serum withdrawal. (a) DCF fluorescence in wild-type NSC34 cells under basal conditions and (b) after serum withdrawal. Scale bar, 50 μm. (c) Serum withdrawal induced a threefold increase in DCF fluorescence in wild-type NSC34 cells. Graph shows means ± 1 SEM. (d) Ebselen reduced serum withdrawal-induced oxidative stress, measured by DCF fluorescence, in NSC34 cells in a dose-dependent manner, with a half-maximal effect (EC50) of 4 μM. There was no increase in toxicity, as measured by EthD1 fluorescence.
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fig2: Oxidative stress can be induced in NSC34 cells by serum withdrawal. (a) DCF fluorescence in wild-type NSC34 cells under basal conditions and (b) after serum withdrawal. Scale bar, 50 μm. (c) Serum withdrawal induced a threefold increase in DCF fluorescence in wild-type NSC34 cells. Graph shows means ± 1 SEM. (d) Ebselen reduced serum withdrawal-induced oxidative stress, measured by DCF fluorescence, in NSC34 cells in a dose-dependent manner, with a half-maximal effect (EC50) of 4 μM. There was no increase in toxicity, as measured by EthD1 fluorescence.

Mentions: To efficiently screen large numbers of potential antioxidant compounds, it was necessary to develop a simple cell-based oxidative stress assay with a large window of effect. Short-term serum withdrawal triggers an increase in oxidative stress in wild-type NSC34 cells [43], which was seen as a threefold increase in DCF fluorescence (Figs. 2a–c). This increase in oxidative stress could be rescued by ebselen (Fig. 2d), an organo-selenium antioxidant compound previously shown to be protective against serum withdrawal-induced death in mSOD1-expressing NSC34 cells [37]. Because a reduction in DCF fluorescence would also occur if ebselen induced cell death through a mechanism not involving oxidative stress, a simultaneous toxicity assay was also incorporated. EthD1 fluoresces when bound to DNA, but is not cell permeable and therefore fluoresces only when bound to DNA in a cell with a compromised membrane. Ebselen reduced oxidative stress measured by DCF fluorescence without increasing EthD1 fluorescence, indicating that the reduced DCF fluorescence was caused by a reduction in oxidative stress (Fig. 2d). This simple assay system could then be used to screen larger numbers of potential antioxidant molecules.


An in vitro screening cascade to identify neuroprotective antioxidants in ALS.

Barber SC, Higginbottom A, Mead RJ, Barber S, Shaw PJ - Free Radic. Biol. Med. (2009)

Oxidative stress can be induced in NSC34 cells by serum withdrawal. (a) DCF fluorescence in wild-type NSC34 cells under basal conditions and (b) after serum withdrawal. Scale bar, 50 μm. (c) Serum withdrawal induced a threefold increase in DCF fluorescence in wild-type NSC34 cells. Graph shows means ± 1 SEM. (d) Ebselen reduced serum withdrawal-induced oxidative stress, measured by DCF fluorescence, in NSC34 cells in a dose-dependent manner, with a half-maximal effect (EC50) of 4 μM. There was no increase in toxicity, as measured by EthD1 fluorescence.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2742740&req=5

fig2: Oxidative stress can be induced in NSC34 cells by serum withdrawal. (a) DCF fluorescence in wild-type NSC34 cells under basal conditions and (b) after serum withdrawal. Scale bar, 50 μm. (c) Serum withdrawal induced a threefold increase in DCF fluorescence in wild-type NSC34 cells. Graph shows means ± 1 SEM. (d) Ebselen reduced serum withdrawal-induced oxidative stress, measured by DCF fluorescence, in NSC34 cells in a dose-dependent manner, with a half-maximal effect (EC50) of 4 μM. There was no increase in toxicity, as measured by EthD1 fluorescence.
Mentions: To efficiently screen large numbers of potential antioxidant compounds, it was necessary to develop a simple cell-based oxidative stress assay with a large window of effect. Short-term serum withdrawal triggers an increase in oxidative stress in wild-type NSC34 cells [43], which was seen as a threefold increase in DCF fluorescence (Figs. 2a–c). This increase in oxidative stress could be rescued by ebselen (Fig. 2d), an organo-selenium antioxidant compound previously shown to be protective against serum withdrawal-induced death in mSOD1-expressing NSC34 cells [37]. Because a reduction in DCF fluorescence would also occur if ebselen induced cell death through a mechanism not involving oxidative stress, a simultaneous toxicity assay was also incorporated. EthD1 fluoresces when bound to DNA, but is not cell permeable and therefore fluoresces only when bound to DNA in a cell with a compromised membrane. Ebselen reduced oxidative stress measured by DCF fluorescence without increasing EthD1 fluorescence, indicating that the reduced DCF fluorescence was caused by a reduction in oxidative stress (Fig. 2d). This simple assay system could then be used to screen larger numbers of potential antioxidant molecules.

Bottom Line: Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system.These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated.Subsequent in vivo studies can be targeted using molecules with the greatest probability of success.

View Article: PubMed Central - PubMed

Affiliation: Academic Neurology Unit and Sheffield Care and Research Centre for Motor Neuron Disorders, University of Sheffield, Sheffield, UK.

ABSTRACT
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, characterized by progressive dysfunction and death of motor neurons. Although evidence for oxidative stress in ALS pathogenesis is well described, antioxidants have generally shown poor efficacy in animal models and human clinical trials. We have developed an in vitro screening cascade to identify antioxidant molecules capable of rescuing NSC34 motor neuron cells expressing an ALS-associated mutation of superoxide dismutase 1. We have tested known antioxidants and screened a library of 2000 small molecules. The library screen identified 164 antioxidant molecules, which were refined to the 9 most promising molecules in subsequent experiments. Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system. The top-performing molecules identified include caffeic acid phenethyl ester, esculetin, and resveratrol. These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated. Subsequent in vivo studies can be targeted using molecules with the greatest probability of success.

Show MeSH
Related in: MedlinePlus